Cytoplasmic Damage and Genotoxicity

细胞质损伤和遗传毒性

• 批准号: 7842077
• 负责人: Tom K. Hei
• 金额: $ 39.34万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-17 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7842077
• 关键词: Address; Alpha Particles; Biological; Biological Assay; Bystander Effect; Cell Nucleus; Cells; Charge; Chromatids; Cytoplasm; Cytosol; DNA; Dose; Dyes; Educational process of instructing; Event; Fibroblasts; Fluorochrome; Funding; Gap Junctions; Generations; Genetic; Glutathione; Hamsters; Human; Hybrids; Incidence; Ionizing radiation; Link; Lipid Peroxidation; Low Dose Radiation; Mammalian Cell; Mediating; Membrane; Membrane Potentials; Mitochondria; Mitochondrial DNA; Mutagenesis; Mutation; Nuclear; Oxidants; Parents; Point Mutation; Process; Proteins; Radiation; Radiobiology; Reactive Oxygen Species; Research Personnel; Respiratory Chain; Risk Assessment; Roentgen Rays; Role; Science; Signal Transduction; Skin; Students; Succinate Dehydrogenase; Universities; cytochrome c oxidase; cytotoxicity; genotoxicity; inhibitor/antagonist; irradiation; mitochondrial DNA mutation; mitochondrial membrane; mutant; oxidative damage; particle; public health relevance; radiation effect; research study; response

DESCRIPTION (provided by applicant): For over a century since the discovery of X-rays by Rventgen, students in radiological sciences have been taught that the quintessential target for radiation induced genetic damages resides in the DNA of the nucleus. The biological consequences of cytoplasmic damage are largely unknown. Using a precision charged particle microbeam and dual fluorochrome dyes to locate nucleus and cellular cytoplasm respectively, thereby avoiding inadvertent traversal of nuclei, the applicant has shown previously that cytoplasmic irradiation is, in fact, mutagenic at the CD59 locus of human-hamster hybrid (AL) cells while inflicting minimal cytotoxicity. With the funding support of the parent R01, we have shown recently that 1) reactive oxygen species mediate this process; 2) targeted cytoplasmic irradiation results in lipid peroxidation of membranes as shown by the induction of 4-hdyroxy-2-nonenal, a major lipid peroxidation byproduct; 3) generated mitochondrial DNA depleted (Ao) AL cells and human fibroblasts; and finally, targeted cytoplasmic irradiation induces bystander mutations and chromatid breaks among neighboring, non-irradiate cells through a gap junction-mediated process. This raised the following questions: What are the effects of targeted cytoplasmic irradiation on mitochondrial DNA mutations and does mitochondrial DNA depleted human fibroblasts demonstrated reduced induction of mutagenesis upon cytoplasmic irradiation? The central hypotheses for this one year supplemental application is targeted cytoplasmic irradiation induces a dose dependent mutagenesis of mitochondrial DNA in human fibroblasts and that mitochondrial DNA depleted cells respond poorly to genotoxic signaling. To address these hypotheses, 2 specific aims are proposed. Mutations will be scored at the HPRT locus in human fibroblasts and mitochondrial DNA mutations (depletion, heteroplasmic deletions and point mutations will be determined. The proposed studies will help to address the mechanisms of how cytoplasmic irradiation results in a genetic event in the nucleus. Together with the bystander mutagenic effect, the study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells. PUBLIC HEALTH RELEVANCE: Generations of students in radiation biology have been taught that heritable biological effects induced by ionizing radiation are the consequence of a direct radiation-nuclear interaction. Using the Columbia University charged particle microbeam and the human hamster hybrid (AL) cell mutagenic assay, there is evidence that targeted cytoplasmic irradiation is mutagenic in mammalian cells. This first, unequivocal demonstration of an extranuclear effect of ionizing radiation provides strong support of the subsequent, broad reaching bystander/ non-targeted effects of radiation. There is evidence that targeted cytoplasmic irradiation can also induce a bystander effect. As such, a better understanding of the genotoxic mechanism of cytoplasmic irradiation is critical in low dose radiation risk assessment. Mitochondria are widely distributed in the cytosol and mitochondrial DNA is highly susceptible to oxidative damage. The objective of the proposed study is to ascertain the possible effects of cytoplasmic irradiation on mitochondrial DNA mutations and the subsequent modulation on mitochondrial function. The study will address some of the fundamental issues regarding extranuclear target and how cytoplasmic damages are being processed in mammalian cells.

描述（由申请人提供）：自Rventgen发现X射线以来的世纪以来，放射科学专业的学生一直被教导辐射诱导遗传损伤的典型靶点位于细胞核的DNA中。细胞质损伤的生物学后果在很大程度上是未知的。使用精确的带电粒子微束和双荧光染料分别定位细胞核和细胞质，从而避免无意中穿过细胞核，申请人先前已经表明，细胞质照射实际上在人-仓鼠杂交（AL）细胞的CD 59基因座处是致突变的，同时造成最小的细胞毒性。在亲本R 01的资助下，我们最近发现：1）活性氧介导了这一过程; 2）靶向细胞质照射导致膜的脂质过氧化，如通过诱导主要的脂质过氧化副产物4-羟基-2-壬烯醛所示; 3）产生线粒体DNA缺失的（Ao）AL细胞和人成纤维细胞;最后，靶向细胞质照射通过间隙连接介导的过程在邻近的未照射细胞中诱导旁观者突变和染色单体断裂。这提出了以下问题：有针对性的细胞质照射对线粒体DNA突变的影响是什么？线粒体DNA耗尽的人成纤维细胞在细胞质照射后是否表现出减少的诱变诱导？这一为期一年的补充申请的中心假设是靶向细胞质辐照诱导人成纤维细胞中线粒体DNA的剂量依赖性诱变，并且线粒体DNA缺失的细胞对遗传毒性信号的反应较差。为了解决这些假设，提出了2个具体目标。将对人成纤维细胞中HPRT基因座的突变进行评分，并确定线粒体DNA突变（耗竭、异质性缺失和点突变）。拟议的研究将有助于解决细胞质照射如何导致细胞核中的遗传事件的机制。与旁观者致突变效应一起，该研究将解决一些关于细胞核靶点以及哺乳动物细胞中细胞质损伤如何处理的基本问题。 公共卫生相关性：几代辐射生物学学生都被教导，电离辐射引起的遗传生物效应是辐射与核直接相互作用的结果。使用哥伦比亚大学带电粒子微束和人仓鼠杂交（AL）细胞致突变试验，有证据表明靶向细胞质照射在哺乳动物细胞中具有致突变性。这是第一次明确证明电离辐射的致核效应，为辐射随后产生的广泛的旁观者/非靶效应提供了强有力的支持。有证据表明，靶向细胞质照射也可以诱导旁观者效应。因此，更好地了解细胞质照射的遗传毒性机制是低剂量辐射风险评估的关键。线粒体广泛分布于细胞质中，线粒体DNA极易受到氧化损伤。该研究的目的是确定细胞质照射对线粒体DNA突变的可能影响以及随后对线粒体功能的调节。这项研究将解决一些关于细胞核靶点的基本问题，以及哺乳动物细胞如何处理细胞质损伤。